DNA analysis by use of a hybridization technique, is a known method (in “Gentechnische Methoden” G. Gassen and G. Schrimpf, Spektrum Akademischer Verlag Heidelberg, 1999, Chapter 11 “Blottingverfahren und Hybridisierungen”, pages 243 to 261). DNA probe molecules, called capture oligonucleotides, which, because of their specific affinity for the complementary sample DNA, “capture” the latter by forming so-called hybrids, i.e. pairs of capture molecule and target molecule, are immobilized on a solid support material. This binding event is normally indicated by optical or else enzymatic reporter molecules.
Such DNA analyses are used for example to detect infectious agents, such as tuberculosis or HIV. DNA analysis must satisfy a particular requirement in the case of so-called single nucleotide polymorphism, SNPs for short. It is necessary here for a capture molecule consisting of about 20 different nucleotides to bind selectively or not bind target molecules which differ in only a single nucleotide. Since the differences in binding energy are very small, the requirements to be met by the selectivity of the DNA sensor are very high.
DNA sensors are known in the art, reference being made for example to the non-prior-published DE 102 59 820 A1 and DE 102 59 821 A1 of the applicant. The formation of the capture/target DNA hybrids takes place under specific boundary conditions, where matching capture/target DNA pairs have a higher binding energy than those having a base mismatch. Because of the small differences in binding energy in SNPs, it is often not possible to distinguish unambiguously a perfect match and a single point mismatch.
A latter problem has to date been solved by introducing a so-called stringent washing step in the prior art analytical methods, i.e. the ionic strength of a washing liquid was chosen so that the initially nonspecifically bound single base mismatch target molecules are separated from the captors, but the perfect match target molecules remain bound to the capture molecules. Even more elaborate optical melting point analyses are likewise possible.
In this method, use is made of the intrinsic change in the light absorption when the DNA double strand melts, and no optical label is necessary. Both with stringent washing and in the optical melting point analysis, in which moreover relatively large amounts of DNA are required and a spectrophotometer is indispensable for detection in liquid phase, it is usually possible for the conditions to be set for only a single SNP. If a plurality of SNPs are present on a sensor chip, separation of all the mismatches is impossible.
In the optical detection of melting curves, the optical signal (intrinsic activity of the DNA, or label) often does not have the stability required for continuous measurements or repeat measurements. The same applies to irreversible detection methods. In particular, it may be necessary for the chip to be dried after the stringent washing before it can be passed on for optical reading.
In “An Active Microelectronics Device for Multiplex DNA Analysis”, M. Heller, IEEE Engineering in Medicine and Biology, March/April 1996, pages 100 to 104 there is further a description of a so-called “electrical stringency treatment” in which the hybridization is carried out on a chip provided with electrodes. Single point mismatch hybrids are separated owing to the polyanionic character of the DNA through a negative polarization of the electrodes.
This method has, however, not been able, to establish itself as a robust and general method. Moreover, accurate knowledge of the respective SNP energy differences is necessary in this method in order to be able to set the individual electrical conditions such as electrical potential, possibly pulse duration and intensity. The DNA may be damaged through the use of high-energy pulses.
In addition, WO 02/083952 Al discloses a method for analyzing nucleic acids in a sample, in which an SNP analysis is carried out and a hybridization of the samples takes place at predetermined temperatures. The analysis in this case takes place in particular by way of the so-called FRET (Fluorescence Resonance Energy Transfer) method. The specification of temperature and setting are said in this case to take place in accordance with U.S. Pat. No. 5,965,410 A, which is cited as integral constituent of WO 02/083952 A1. In particular, a specific program with which, when the flow is stationary, the temperature can be set at a defined previously calculated value is indicated therein.
In addition, U.S. Pat. No. 6,391,558 A discloses an electrochemical method for detecting DNA, in which a quantitatively readable electrical signal is generated in the hybridization process.